Mutant proteases and uses thereof

Abstract

The present disclosure, in some aspects, provides mutant TEV proteases that exhibit improved activity, where the mutant TEV exhibits increase efficiency and/or an increased Kcat for cleavage of an amino acid sequence, and TEV proteases are commonly used for laboratory methods including cleaving fusion proteins and removing a purification tag, such as a maltose binding protein or a poly-histidine tag, from a fusion protein or an antibody.

1 . A polypeptide comprising an active mutant TEV (Tobacco Etch Virus) protease, wherein the mutant TEV protease comprises:
 (i) substitution mutations at amino acid residues corresponding to amino acids S3, P8, S31, and A231 of SEQ ID NO:1; and/or   (ii) a substitution mutation of arginine at amino acid residue corresponding to S219 of SEQ ID NO:1.   
     
     
         2 . The polypeptide of  claim 1 , wherein the mutant TEV protease further comprises a substitution mutation at amino acid residue corresponding to amino acid T173 of SEQ ID NO:1. 
     
     
         3 . The polypeptide of  claim 1 , wherein the active mutant TEV protease comprises alanine at position 173 (T173A). 
     
     
         4 . The polypeptide of  claim 1 , wherein the mutant TEV protease comprises substitution mutations of: isoleucine at position 3 (S31), glutamine at position 8 (P8Q), threonine at position 31 (S31T), and a substitution mutation at position 231. 
     
     
         5 . The polypeptide of  claim 4  wherein, the mutant TEV protease comprises valine at position 231 (A231V). 
     
     
         6 . (canceled) 
     
     
         7 . The polypeptide of  claim 4 , wherein the mutant TEV protease comprises the substitution mutation of S219R. 
     
     
         8 . The polypeptide of  claim 4 , wherein the mutant TEV protease comprises the substitution mutations of S3I, P8Q, S31T, T173A, and A231V. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The polypeptide of  claim 8 , wherein the mutant TEV protease comprises the substitution mutations of S3I, P8Q, S31T, T173A, S219R, and A231V. 
     
     
         13 . The polypeptide of  claim 1 , wherein the polypeptide comprises or consists of SEQ ID NO:3, SEQ ID NO:4, or a sequence having at least 95% sequence identity thereto. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The polypeptide of claim  15 , wherein the mutant TEV protease has an amino acid sequence at least 95% identical to (SEQ ID NO:1), and wherein the mutant TEV protease comprises substitution mutations at the amino acid residues corresponding to positions S3I, P8Q, S31T, T173A, and A231V of (SEQ ID NO:1). 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The polypeptide of  claim 1 , wherein the polypeptide can cleave Glu-X-X-Tyr-X-Gln/Xa, (SEQ ID NO:5) at a rate that is faster than the wild-type TEV protease. 
     
     
         20 . The polypeptide of  claim 19 , wherein the polypeptide can cleave ENLYFQG (SEQ ID NO:6) or ENLYFQS (SEQ ID NO:7) at a rate of greater than a k cat  value of 0.17 s −1 . 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The polypeptide of  claim 1 , wherein the polypeptide further comprises a polypeptide tag sequence. 
     
     
         25 . The polypeptide of  claim 24 , wherein the polypeptide tag sequence is a metal binding tag, a histidine tag or polyhistidine tag (His-tag), a HQ tag, a glutathione S-transferase (GST) tag, a N-Utilization substance (NusA) tag, thioredoxin (TRX), trigger factor, SUMO, or a polyarginine tag. 
     
     
         26 . The polypeptide of  claim 1 , wherein the polypeptide is covalently bound to a maltose binding protein (MBP). 
     
     
         27 - 28 . (canceled) 
     
     
         29 . The polypeptide of  claim 1 , wherein the polypeptide comprises a polyhistidine tag or a polyarginine tag. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . The polypeptide of  claim 29 , wherein the polypeptide comprises an N-terminal polyhistidine tag and a C-terminal polyarginine tag. 
     
     
         33 - 36 . (canceled) 
     
     
         37 . The polypeptide of  claim 1 , wherein the polypeptide is covalently attached to or expressed as a fusion protein with a fluorescent protein. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . The polypeptide of  claim 1 , wherein the mutant TEV protease comprises SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:18. 
     
     
         41 . A method of cleaving an amino acid bond in a polypeptide comprising contacting the polypeptide with a mutant protease of  claim 1 . 
     
     
         42 - 50 . (canceled) 
     
     
         51 . A kit comprising the polypeptide of  claim 1  and a suitable container. 
     
     
         52 - 54 . (canceled)